title: Thermostable variants of Zymomonas mobilis alcohol dehydrogenase obtained using PCR-mediated random mutagenesis.
abstract: Using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant over 10 degrees C more stable at pH 8, with essentially unaltered kinetic characteristics, was produced. However, the pH profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees C) at pH 6. Sequencing of the variants indicated five amino acids changes which contributed to thermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were contained in the final stable variant, and the changes were partially additive, with individual mutations causing between 2 and 3.5 degrees C stability increases (at pH 7.5). It is estimated that the most stable variant would have a half-life under physiological conditions at 70 degrees C of 15 min.
authors.affiliation: School of Biochemistry, La Trobe University, Bundoora, Melbourne, Victoria, 3083, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Rellos
authors.forename: L
authors.initials: L
authors.lastname: Pinheiro
authors.forename: R K
authors.initials: RK
authors.lastname: Scopes
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013696:Temperature; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics*
chemical_list: D000426:Alcohol Dehydrogenase